Interferon (IFN) plays a central role in the innate and adaptive antiviral immune responses. While IFN-alfa is currently approved for treating chronic hepatitis B and hepatitis C, IFN-gamma, in limited studies, has not been shown to be effective for chronic hepatitis B or C. To identify the potential mechanism underlying the differential antiviral effects of IFN-alfa and IFN-gamma, we used cDNA microarray to profile the global transcriptional response to IFN-alfa and IFN-gamma in primary human hepatocytes, the target cell population of hepatitis viruses. Our results reveal distinct patterns of gene expression induced by these two cytokines. Overall, IFN-alfa induces more genes than IFN-gamma at the transcriptional level. Distinct sets of genes were induced by IFN-alfa and IFN-gamma with limited overlaps. IFN-alfa induces gene transcription at an early time point (6 hours) but not a later time point (18 hours), while the effects of IFN-gamma are more prominent at 18 hours than 6 hours, suggesting a delayed transcriptional response to IFN-gamma in the hepatocytes. These findings indicate differential actions of IFN-alfa and IFN-gamma in the context of therapeutic intervention for chronic viral infections in the liver. The combination of peginterferon and ribavirin is the standard treatment for chronic hepatitis C. Data from our recent clinical study suggests that ribavirin augments the induction of interferon stimulated genes (ISGs) in patients treated for HCV infection. In order to further characterize the mechanisms of action of ribavirin in combination therapy, we examined the effect of ribavirin treatment on ISG induction in mammalian cells grown in tissue culture. In addition, the effect of ribavirin on infectious HCV cell culture systems was also studied. In order to further characterize the mechanisms of action of ribavirin, we examined the effect of ribavirin treatment on ISG induction in cell culture. In addition, the effect of ribavirin on infectious HCV cell culture systems was also studied. Similar to interferon-&#945;, ribavirin potently inhibits JFH-1 infection of Huh7.5.1 cells in a dose-dependent manner, which spans the physiological concentration of ribavirin in vivo. Microarray analysis and subsequent quantitative PCR assays demonstrated that ribavirin treatment resulted in the induction of a distinct set of ISGs. These ISGs, including IRF7 and IRF9 are known to play an important role in anti-HCV responses. When ribavirin is used in conjunction with interferon, induction of specific ISGs is synergistic when compared to either drug applied separately. Direct up-regulation of these antiviral genes by ribavirin is mediated by a novel mechanism different from those associated with interferon signaling and intracellular double stranded RNA sensing pathways such as RIG-I and MDA5. RNA interference studies excluded the activation of the Toll-like receptor and NF-KappaB pathways in the action of ribavirin. In conclusion, our study suggests that ribavirin, acting via a novel innate mechanism, potentiates the anti-HCV effect of interferon. Understanding the mechanism of action of ribavirin would be valuable in identifying novel antivirals. To further understand the mechanisms of ribavirin action and interferon response in HCV therapy, we studied early viral kinetics, serum cytokine expression and viral mutagenesis during peginterferon treatment with and without ribavirin. 50 patients with chronic hepatitis C virus (HCV) infection genotype 1 were randomized to receive peginterferon alfa-2a with or without ribavirin for the 1st month of treatment. All patients then received 48 weeks of combination therapy. First and second phase viral kinetics were evaluated. Serum IP10, MIG and MCP1 levels, representative of ISG response, were measured. Sequencing of part of NS5A and NS5B was performed and mutation rates calculated. First phase decline was similar between groups. Patients receiving ribavirin had more rapid second phase kinetics, particularly those with an adequate first phase decline (0.61 vs 0.35 log10 IU/mL/week, p=0.018). Induction of serum IP10 at 12 hrs was higher in patients on ribavirin than those receiving peginterferon alone (7.6 vs 3.8 fold, p=0.01);however, the difference was more apparent in those with an adequate first phase decline. IP10-induction correlated with 1st and 2nd phase kinetics and correlated with day 3 ribavirin serum concentration. Sequencing analysis revealed similar mutation rates in both groups. Ribavirin improves early viral kinetics, but only in patients with an adequate initial response to peginterferon. Greater induction of interferon-stimulated cytokines and the correlation with viral kinetics in ribavirin-treated patients, support the notion that ribavirin may act through augmentation of interferon signaling S-adenosyl methionine (SAMe) has been shown to enhance interferon signaling in cell culture and animal models by acting as a methyl donor to STAT1, leading to improved STAT1-DNA binding. We performed a clinical study to assess the effect of SAMe on interferon signaling and early viral kinetics in previous non-responders to peginterferon and ribavirin. Previous genotype-1 non-responders were given 2 weeks of peginterferon alfa-2a and ribavirin to establish baseline responses (Course A). After a 1-month washout, patients received SAMe 1600mg daily for 2 weeks, whereupon peginterferon and ribavirin were added for 48 weeks (Course B). Viral kinetics and interferon stimulated gene (ISG) expression in PBMCs were compared between courses. 21 of 24 patients completed both Course A and B, serving as their own controls. HCV RNA decline from 0 to 48 hours (phase 1) was similar with and without SAMe. In contrast, the second phase slope of viral decline was improved with SAMe (Course A 0.110.04 vs Course B 0.270.06 log10IU/mL/week, p=0.009). 11 (53%) patients achieved an early virological response and 10 (48%) had undetectable HCV RNA by week 24. Induction of ISGs in PBMCs was significantly greater in Course B. Cell culture data confirmed that SAMe augmented ISG induction and antiviral effects of interferon by increasing STAT1 methylation, possibly affecting STAT1-DNA binding. The addition of SAMe improves early viral kinetics and ISG induction in interferon non-responders. SAMe holds promise as an adjunct to peginterferon-based therapy in chronic HCV infection.